Cks1 mediates vascular smooth muscle cell polyploidization

Vascular smooth muscle cells (VSMC) at capacitance arteries of hypertensive individuals and animals undergo dramatic polyploidization that contributes toward their hypertrophic phenotype. We report here the identification of a defective mitotic spindle cell cycle checkpoint in VSMC isolated from capacitance arteries of pre-hypertensive rats. These cells demonstrated a high predisposition to polyploidization in culture and failed to maintain cyclin B protein levels in response to colcemid, a mitotic inhibitor. Furthermore, this altered mitotic spindle checkpoint status was associated with the overexpression of Cks1, a Cdc2 adapter protein that promotes cyclin B degradation. Cks1 up-regulation, cyclin B down-regulation, and VSMC polyploidization were evidenced at the smooth muscle of capacitance arteries of genetically hypertensive and Goldblatt-operated rats. In addition, angiotensin II infusion dramatically increased Cks1 protein levels at capacitance arteries of normotensive rats, and angiotensin II treatment of isolated VSMC abrogated their ability to down-regulate Cks1 and maintain cyclin B protein expression in response to colcemid. Finally, transduction of VSMC from normotensive animals with a retrovirus that drives the expression of Cks1 was sufficient to alter their mitotic spindle cell cycle checkpoint status and promote unscheduled cyclin B metabolism, cell cycle re-entry, and polyploidization. These data demonstrate that Cks1 regulates cyclin B metabolism and ploidy in VSMC and may contribute to the understanding of the phenomena of VSMC polyploidization during hypertension.     

Nicotine-induced vascular endothelial growth factor release via the EGFR-ERK pathway in rat vascular smooth muscle cells

Cigarette smoke has been firmly established as an independent risk factor for atherosclerosis and other vascular diseases. The proliferation and migration of vascular smooth muscle cells (VSMC) induced by growth factors have been proposed to play an important role in the progression of atherosclerosis. In the present study, we investigated the effects of nicotine, which is one of the important constituents of cigarette smoke, on vascular endothelial growth factor (VEGF) release, in rat VSMC. The stimulation of cells with nicotine resulted in a time- and concentration-dependent release of VEGF. Hexamethonium, an antagonist of nicotinic acetylcholine receptor (nAChR), inhibited nicotine-induced VEGF release. We next investigated the mechanisms by which nicotine induces VEGF release in the cells. The nicotine-induced VEGF release was inhibited by treatment with U0126, a selective inhibitor of MEK, which attenuated the nicotine-induced ERK phosphorylation. Nicotine induced a transient phosphorylation of ERK. Furthermore, AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) kinase, inhibited nicotine-induced ERK phosphorylation and VEGF release. These data suggest that nicotine releases VEGF through nAChR in VSMC. Moreover, VEGF release induced by nicotine is mediated by an EGFR-ERK pathway in VSMC. VEGF may contribute to the risk of cardiovascular diseases in cigarette smokers.     

Tumor necrosis factor alpha inhibits insulin-induced mitogenic signaling in vascular smooth muscle cells

Tumor necrosis factor alpha (TNFalpha) interferes with insulin signaling in adipose tissue and may promote insulin resistance. Insulin binding to the insulin receptor (IR) triggers its autophosphorylation, resulting in phosphorylation of Shc and the downstream activation of p42/p44 extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2), which mediates insulin-induced proliferation in vascular smooth muscle cells (VSMC). Since insulin resistance is a risk factor for vascular disease, we examined the effects of TNFalpha on mitogenic signaling by insulin. In rat aortic VSMC, insulin induced rapid phosphorylation of the IR and Shc and caused a 5.3-fold increase in activated, phosphorylated ERK1/2 at 10 min. Insulin induced a biphasic ERK1/2 activation with a transient peak at 10 min and a sustained late phase after 2 h. Preincubation (30-120 min) with TNFalpha had no effect on insulin-induced IR phosphorylation. In contrast, TNFalpha transiently suppressed insulin-induced ERK1/2 activation. Insulin-induced phosphorylation of Shc was inhibited by TNFalpha in a similar pattern. Since mitogenic signaling by insulin in VSMC requires ERK1/2 activation, we examined the effect of TNFalpha on insulin-induced proliferation. Insulin alone induced a 3.4-fold increase in DNA synthesis, which TNFalpha inhibited by 48%. TNFalpha alone was not mitogenic. Inhibition of ERK1/2 activation with PD98059 also inhibited insulin-stimulated DNA synthesis by 57%. TNFalpha did not inhibit platelet-derived growth factor-induced ERK1/2 activation or DNA synthesis in VSMC. Thus, TNFalpha selectively interferes with insulin-induced mitogenic signaling by inhibiting the phosphorylation of Shc and the downstream activation of ERK1/2.     

Epidermal growth factor induction of phenotype-dependent cell cycle arrest in vascular smooth muscle cells is through the mitogen-activated protein kinase pathway

The heterogeneity of vascular smooth muscle cells is well established in tissue culture, but their differential responses to growth factors are not completely defined. We wished to identify effects of epidermal growth factor (EGF) on vascular smooth muscle cells in distinct phenotypes, such as spindle and epithelioid. We found that the EGF receptors were abundant in epithelioid cells but not spindle cells. EGF treatment inhibited serum-independent DNA synthesis, which was absent in spindle cells, of epithelioid cells. Additionally, using a pulse-chase assay, we found that bromodeoxyuridine-labeled cells failed to re-enter the S phase in the presence of EGF. These EGF effects were abolished by either inhibiting the EGF receptor tyrosine kinase with AG1478 or inhibiting the mitogen-activated protein kinase pathway with PD98059. In response to treatment with EGF, the EGF receptor was phosphorylated, which was correlated with phosphorylation and activation of p42/44 mitogen-activated protein kinases. Inhibition of EGF receptor phosphorylation and mitogen-activated protein kinase activation resulted in a reversal of the EGF-induced inhibition of bromodeoxyuridine incorporation and cell cycle arrest. Subsequent studies revealed that the activation of the EGF receptor and the mitogen-activated protein kinase pathway in epithelioid cells induced expression of the cell cycle inhibitory protein p27Kip1 but not p21Cip1. Taken together, our data demonstrate that the EGF receptor is abundantly expressed in epithelioid vascular smooth muscle cells and that the activation of this receptor results in cell cycle arrest through activation of the mitogen-activated protein kinase pathway.     

Linkage between alpha(1) adrenergic receptor and the Jak/STAT signaling pathway in vascular smooth muscle cells

The Jak/STAT pathway is activated following stimulation of the type I angiotensin II receptor. To examine whether this pathway is shared among other G-protein-coupled receptors, we studied the linkage between the alpha(1) adrenergic receptor and this pathway. The alpha(1) agonist phenylephrine induced tyrosine phosphorylation of Jak2, Tyk2, and STAT1 in vascular smooth muscle cells. The phosphorylation of Jak2 was prevented by the alpha(1) receptor antagonists prazosin and chloroethylclonidine, but not by WB4101, and that of STAT1 was inhibited by prazosin and the Jak2 inhibitor AG490. After stimulation with phenylephrine, Jak2 and STAT1 were found to associate with alpha(1B) receptor. Phenylephrine stimulated the DNA binding activity of STAT1. Protein synthesis promoted by phenylephrine was inhibited by prazosin, AG490, and the introduction of a decoy oligonucleotide for STAT1. These results suggested that alpha(1) receptor is linked to the Jak/STAT pathway and that this pathway mediates alpha(1) agonist-induced smooth muscle hypertrophy.     

Modulation of collagen synthesis by tumor necrosis factor alpha in cultured vascular smooth muscle cells

Collagen synthesis in vascular smooth muscle cells (SMCs) after exposure to tumor necrosis factor alpha (TNF-alpha) was investigated using a culture system. The synthesis of collagenase-digestible proteins (CDP) and noncollagenous proteins (NCP) was evaluated by the [3H]proline incorporation. It was shown that TNF-alpha markedly suppresses the incorporation of [3H]proline into both CDP and NCP in confluent cultures of SMCs but not in sparse cultures of the cells. Such a marked suppression by TNF-alpha was not observed in confluent bovine aortic endothelial cells and human fibroblastic IMR-90 cells. In confluent SMCs, the synthesis of CDP was more strongly inhibited by TNF-alpha than that of NCP. When the CDP synthesis was stimulated by transforming growth factor beta, TNF-alpha suppressed the stimulation in both confluent and sparse SMCs. Human SMCs synthesized types I, III, IV and V collagens; TNF-alpha markedly decreased the relative proportion of types IV and V. It was therefore suggested that TNF-alpha modulates the collagen synthesis by SMCs depending on their cell density and modifies the formation of atherosclerotic lesions.     

Impact of the tissue factor pathway inhibitor gene on apoptosis in human vascular smooth muscle cells

Tissue factor pathway inhibitor (TFPI) plays a vitally important role in the blood coagulation pathway. Recent studies indicated that TFPI induces apoptosis in vascular smooth-muscle cells (VSMCs) in animals. The present study investigated whether the TFPI gene could also induce apoptosis in human vascular smooth-muscle cells (hVSMCs). Such cells were isolated from human umbilical arteries and subsequently transfected with pIRES-TFPI plasmid (2 μg/mL). MTT assaying and cell counting were applied to measure cell viability and proliferation, RT-PCR was utilized to analyze TFPI gene expression in the cells. Apoptosis was analyzed by fluorescence activated cell sorting (FACS). Several key proteins involved in apoptosis were examined through Western blotting. It was shown that TFPI gene transfer led to its increased cellular expression, with a subsequent reduction in hVSMC proliferation. Further investigation demonstrated that TFPI gene expression resulted in lesser amounts of procaspase-3, procaspase-8 and procascase-9, and an increased release of mitochondrial cytochrome c (cyt-c) into cytoplasm, thereby implying the involvement of both extrinsic and intrinsic pathways in TFPI gene-induced apoptosis in hVSMCs.     

Autocrine thrombospondin partially mediates TGF-beta1- induced proliferation of vascular smooth muscle cells

Transforming growth factor (TGF)-beta1 has been implicated in vascular healing responses after mechanical injury. Using cultured rat aortic smooth muscle cells (RASMC), we examined the hypothesis that production and secretion of thrombospondin (TSP) contributes to TGF-beta1-induced proliferation. We found that TGF-beta1 enhanced production and secretion of TSP, with peak levels of secreted TSP observed 24 h after treatment. RASMC treated with TGF-beta1 secreted a mitogenic activity that was transferable in conditioned media and partially inhibited by C6.7, a monoclonal anti-TSP antibody. Exogenous TSP stimulated a proliferative response, with maximal [(3)H]thymidine incorporation occurring 24 h earlier than maximal [(3)H]thymidine incorporation in response to TGF-beta1-treatment. Pretreatment with C6.7 or polyclonal anti-TSP neutralizing antibodies inhibited TGF-beta1-induced proliferation of RASMC. Proliferative responses to TGF-beta1 were also inhibited by pretreatment with an anti-beta(3) integrin monoclonal blocking antibody (F11), RGD peptides, and the anti-alpha(v)beta(3) disintegrin echistatin. Treatment with TSP and TGF-beta1 increased c-Jun NH(2)-terminal kinase (JNK)1 activity, with peak effects observed at 15 min and 4 h, respectively. Treatment with C6.7 or F11 inhibited TGF-beta-induced activation of JNK1. In summary, these studies support the hypothesis that TGF-beta-induced JNK1 activation and proliferation of RASMC require secretion of TSP and ligation of alpha(v)beta(3)-integrins.     

Adenovirus-mediated gene transfer of tissue factor pathway inhibitor induces apoptosis in vascular smooth muscle cells

Objective To investigate the pro-apoptotic effect of tissue factor pathway inhibitor (TFPI) gene transfer mediated by adenovirus on vascular smooth muscle cells (VSMCs). Methods Rat VSMCs were infected with recombinant adenovirus containing either the TFPI (Ad-TFPI) or LacZ (Ad-LacZ) gene or DMEM in vitro. TFPI expression was detected by ELISA. Apoptosis of VSMCs was determined by electron microscopy and flow cytometry. The expression of cytochrome c, procaspase-3, cleaved caspase-3, cleaved caspase-9 and inhibitor of apoptosis protein-1(IAP-1) were examined by western blot and RT-PCR. Results TFPI protein was detected in the TFPI group after gene transfer and the peak expression was at the 3rd day. At the 3rd, 5th and 7th day after gene transfer, the apoptotic rates in the TFPI group were 11.95%, 71.96% and 37.83%, respectively, whereas those in the LacZ group were 1.34%, 1.83% and 6.37%, respectively. We observed cell contraction, slight mitochondrial swelling, nuclear pyknosis and apoptotic body formation in TFPI-treated VSMCs using electron microscopy. Cytochrome c, cleaved caspase-3 and cleaved caspase-9, which are all involved in mitochondrial pathway, were detected in the cytoplasm on the 3rd, 5th and 7th day after TFPI gene transfer. Procaspase-3 expression was significantly decreased over time in the TFPI group (each P < 0.05), which were not seen in the Ad-LacZ and DMEM groups. The expression of IAP-1 mRNA in the TFPI group was also decreased compared with the Ad-LacZ and DMEM groups (each P < 0.05) at the 3rd and 7th day after gene transfer. Conclusion The results demonstrated that overexpression of TFPI gene might induce VSMC apoptosis in vitro through the mitochondrial pathway; meanwhile, IAP-1 expression is decreased. These findings indicated that TFPI might inhibit restenosis by inducing apoptosis in VSMCs.     

Receptor-dependent prorenin activation and induction of PAI-1 expression in vascular smooth muscle cells

Although elevated plasma prorenin levels are commonly found in diabetic patients and correlate with microvascular complications, the pathological role of these increases, if any, remains unclear. Prorenin/renin binding to the prorenin/renin receptor [(p)RR] enhances the efficiency of angiotensinogen cleavage by renin and unmasks prorenin catalytic activity. We asked whether plasma prorenin could be activated in local vascular tissue through receptor binding. Immunohistochemical staining showing localization of the (p)RR in the aorta to vascular smooth muscle cells (VSMCs). After cultured rat VSMCs were incubated with 10(-7) M inactive prorenin, cultured supernatant acquired the ability to generate ANG I from angiotensinogen, indicating that prorenin had been activated. Activated prorenin facilitated angiotensin generation in cultured VSMCs when exogenous angiotensinogen was added. Small interfering RNA (siRNA) against the (p)RR blocked this activation and subsequent angiotensin generation. Prorenin alone induced dose- and time-dependent increases in mRNA and protein for the profibrotic molecule plasminogen activator inhibitor (PAI)-1, effects that were blocked by siRNA, but not by the ANG II receptor antagonist saralasin. When inactive prorenin and angiotensinogen were incubated with cells, PAI-1 mRNA increased a striking 54-fold, 8-fold higher than the increase seen with prorenin alone. PAI-1 protein increased 2.75-fold. These effects were blocked by treatment with siRNA + saralasin. We conclude that prorenin at high concentration binds the (p)RR on VSMCs and is activated. This activation leads to increased expression of PAI-1 via ANG II-independent and -dependent mechanisms. These data provide a mechanism by which elevated prorenin levels in diabetes may contribute to the progression of fibrotic disease.